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目的探讨胃泌素对结肠癌细胞CoLo320WT中粘着斑激酶(FAK)通路下游E-钙粘蛋白/β-连环蛋白(E-cadherin/β-catenin)复合物分布的影响；方法脂质体转染表达胃泌素受体CCK-2R的pCR3．1/GR质粒于结肠癌细胞CoLo320中,G418筛选出稳定表达CCK-2R的阳性克隆,RT-PCR鉴定,转染成功命为CoLo320WT。应用10~(-8)mmol/L胃泌素(G17)以时间梯度(0h、1h、6h、12h、24h、48h)干预CoLo320WT细胞,同时应用10~(-6)mmol/L胃泌素受体拮抗剂L365．260干预CoLo320WT细胞30 min,再予10~(-8)mmol/L胃泌素干预。采用免疫印迹法检测磷酸化的FAK Tyr397和总FAK的表达。采用免疫共沉淀和免疫印迹法检测CoLo320WT中TX-100溶解和未溶部分中的E-钙粘蛋白和β-连环蛋白的表达。用免疫细胞化学法观察E-钙粘蛋白和β-连环蛋白的在胞膜、胞质和胞核的分布。结果随着胃泌素干预时间的延长,细胞中磷酸化的FAK Tyr397的表达量呈增加趋势,12h达最大值。胃泌素受体拮抗剂L365,260阻断后磷酸化的FAK Tyr397表达减少。而胃泌素对总FAK没有明显影响。TX-100可溶性部分中E-钙粘蛋白和β-连环蛋白的量在胃泌素干预后表达减少,拮抗剂L365,260阻断后又增加。而TX- 100不溶解部分中表达却相反。免疫细胞化学观察到在胃泌素干预下CoLo320WT细胞中E-钙粘蛋白和β-连环蛋白的分布发生胞质和胞核转移。结论胃泌素与其受体CCK-2受体结合,磷酸化的FAK Tyr397、激活FAK通路进而影响结肠癌细胞中E-钙粘蛋白和β-连环蛋白的分布,促进结肠癌细胞侵袭和转移。
Objective To investigate the effect of gastrin on the distribution of the E-cadherin/β-catenin complex downstream of the focal adhesion kinase (FAK) pathway in colon cancer cells CoLo320WT; Methods Liposomal transfection The pCR3.1/GR plasmid expressing gastrin receptor CCK-2R was detected in colon cancer cell line CoLo320. A positive clone stably expressing CCK-2R was selected by G418. RT-PCR was used to identify the clone. The transfection success was CoLo320WT. 10 ~ (-8) mmol/L gastrin (G17) was used to intervene CoLo320 WT cells with time gradients (0h, 1h, 6h, 12h, 24h, 48h), while 10 ~ (-6) mmol/L gastrin was applied simultaneously. Receptor antagonist L365.260 interfered with CoLo320WT cells for 30 min, and then received 10~(-8) mmol/L gastrin intervention. The expression of phosphorylated FAK Tyr397 and total FAK was detected by Western blotting. Co-immunoprecipitation and Western blot were used to detect the expression of E-cadherin and β-catenin in the soluble and insoluble fractions of TX-100 in CoLo320WT. The distribution of E-cadherin and β-catenin in the cell membrane, cytoplasm, and nucleus was observed by immunocytochemistry. Results With the prolongation of gastrin intervention time, the expression of phosphorylated FAK Tyr397 increased in cells and reached the maximum at 12h. The gastrin receptor antagonist L365,260 blocks post-phosphorylated FAK Tyr397 expression. Gastrin did not have a significant effect on total FAK. The amount of E-cadherin and β-catenin in the soluble fraction of TX-100 was reduced after gastrin intervention and increased after blockade of L365,260. However, the expression in the insoluble portion of TX-100 is reversed. Immunocytochemistry observed cytoplasmic and nuclear translocation of E-cadherin and β-catenin in CoLo320WT cells under gastrin intervention. Conclusions Gastrin binds to its receptor CCK-2 receptor, phosphorylated FAK Tyr397, activates FAK pathway and affects the distribution of E-cadherin and β-catenin in colon cancer cells, and promotes invasion and metastasis of colon cancer cells.